ADP-ribosylation factors, or ARFs, are 20 kDa guanine nucleotide-binding proteins that were first identified by their ability to enhance cholera toxin-catalyzed ADP-ribosylation of Gsalpha and have recently been shown to participate in protein trafficking through the Golgi. GTP-binding proteins, including ARFs, participate in a highly regulated cycle of activation and inactivation. This is accomplished by binding GTP (activation) and its subsequent hydrolysis to GDP (inactivation). The mechanisms underlying this cycle are unknown but are important to understanding the function of the ARF proteins. Since ARF possesses no intrinsic GTPase activity, it may rely on other cellular factors for participation in the cycle. To understand better these mechanisms, we have investigated the nucleotides bound to specific ARF proteins that were synthesized in Escherichia coli under different conditions and correlated these with the activation state of the protein. One of the ARF proteins, human ARF 6, when expressed as a fusion protein, was isolated in an activated state which was independent of exogenous GTP. Activation was due to the presence of bound GTP and its removal resulted in restoration of GTP-dependent activity. By contrast, recombinant ARF 6 expressed as a nonfusion protein was isolated in an inactive state due to the presence of bound GDP. Thus, the presence of the fusion protein appeared to protect the gamma-phosphate of bound GTP from hydrolysis resulting in purification of an activated protein. Cholera toxin ADP-ribosyltransferase activity exhibits an optimal temperature significantly below that of body core temperature (approximately 37 degrees C) found in the jejunum, its site of action in disease. In the presence of phospholipids and a soluble brain ARF, activity was enhanced over a wide range of temperatures; in particular, activity was stimulated at 37 degrees C. ARF thus may enable the organism to adapt to environmental conditions by stimulating toxin transferase activity at physiological temperatures.